The Effect Of NETosis Induced By Antiphospholipid Antibodies On The Function Of Trophoblasts And Endothelial Cells

Backgrounds and ObjectivesThe main clinical history of antiphospholipid syndrome(APS)includes thrombosis and/or pregnancy morbidity.Patients with APS who had clinical history of adverse pregnancy outcomes but without a history of thrombosis are called obstetric APS(OAPS).According to the 2006 Sydney international consensus statement on an update of the classification criteria for definite antiphospholipid syndrome,the pregnancy morbidity included:recurrent miscarriage before 10 weeks of pregnancy,fetal loss after 10 weeks of pregnancy,fetal growth restriction,preeclampsia or premature birth due to placental insufficiency.Laboratory diagnosis includes two or more times high titer in anticardiolipin antibody(aCL)or anti-?2 glycoprotein I antibodies(a(?2GPI)IgM and/or IgG,or lupus Lupus anticoagulant(LA)was positive,and the interval between two tests was at least 12 weeks.At present,the conventional treatment for OAPS is low-dose aspirin combined with preventive-dose low-molecular-weight heparin.More than 80%of patients with OAPS could have a live birth,but some patients have increased risk of pregnancy complications,which suggests that the pathogenesis of OAPS needs further exploration.NETosis is one way in which neutrophils are activated,which described the formation process of neutrophil extracellular traps(NETs).NETs were first discovered in infectious diseases.Recently,the relationship between NETs and non-infectious diseases is emerging.NETs are mainly composed of DNA backbone and various proteases attached to it,including myeloperoxidase(MPO),neutrophil elastase(NE),cathepsin G,et al.The contents of NETs may be different according to different stimuli,and different contents have different biological functions.The molecular mechanisms that regulate the release of NETs are diverse and vary with different stimuli.Many studies have shown that the release of NETs depends on respiratory bursts and is related to intracellular signaling pathways such as Raf/MEK/ERK.The PI3K/AKT pathway has been shown to play an important role in the stimulation of NETs formation by specific immune complexes.Research by Hiromichi et al.showed that inhibition of PI3K down-regulates AKT and Raf/MEK/ERK pathways simultaneously.In addition,Thiago and others believe that the activation of PI3K,AKT and its downstream ERK is very important for NETs generated by reactive oxygen species(ROS).The RNA-LL37 complex in NETs can promote the release of cytokines and chemokines by Toll-like receptor(TLR)8 and can activate neutrophils to release more NETs,which plays an important role in the pathogenesis of psoriasis.In systemic lupus erythematosus(SLE),the DNA-LL37 complex from NETs activates polyclonal B cells through TLR9 to promote their release of autoantibodies.Autoantibodies and antigens form immune complexes and deposite in peripheral tissues to cause harmful inflammatory reactions.In atherosclerotic diseases,cholesterol promotes the release of NETs,which in turn cause macrophages to release cytokines such as interleukin 1?(IL-1?)and activate Th 17 cells.A large number of neutrophils were found in the placenta of pregnancies with obstetric complications such as preeclampsia and fetal growth restriction caused by antiphospholipid antibodies.After neutrophils were eliminated from the body,the antiphospholipid antibody-mediated obstetric complications no longer occurred.In the placenta of patients with SLE and/or preeclampsia,a large number of NETs infiltrate the extravillous space.Placental formation disorder is the main cause of obstetric complications such as recurrent miscarriage,preeclampsia,and fetal growth restriction.The normal functions of trophoblasts and endothelial cells are critical to placental formation.Antiphospholipid antibodies can reduce the ability of trophoblasts to migrate and invade,and can also impair endothelial migration and angiogenesis.The mechanism of their occurrence is unclear.Therefore,this study intends to observe the effect of antiphospholipid antibodies on NETs production by detecting changes of NETs levels in the serum of patients with OAPS in early pregnancy,and then explore the molecular mechanism of antiphospholipid antibodies-stimulated NETs release,and explore the effects of antiphospholipid antibody-induced NETs on trophoblast and endothelial cells.Methods1.Sample collectionFresh peripheral blood from 22 patients with obstetric antiphospholipid syndrome in early pregnancy and 22 normal pregnant women with matching gestational weeks were from the Shandong Provincial Hospital Affiliated to Shandong University.All volunteers obtained informed consent and the study was approved by the hospital ethics committee.2.Detection of NETs markers in serumQuant-iTTM PicoGreen(?)dsDNA Reagent and Kits were used to detect the levels of cell-free DNA in serum.The levels of NETs markers such as MPO-DNA,MPO,NE in serum were detected using ELISA assay.3.Isolation of human peripheral blood neutrophilsPolymorphprep TM separation solution was used to isolate human peripheral blood neutrophils,and trypan blue staining was used to detect neutrophil activity.4.NETs release experimentQuant-iTTM PicoGreen(?)dsDNA Reagent and Kits and live-cell fluorescence staining were used to detect the levels of NETs released by neutrophils in early pregnancy patients with OAPS and normal pregnant women in unstimulated state5.Purification of IgG from human serumThe IgG in serum was separated and purified by NAb TM Protein A Plus Spin Kit,and the protein concentration was detected by BCA assay6.Effect of antiphospholipid antibodies on NETs release15 ?g/mL purified IgG(APS-IgG)in the serum of OAPS patients in early pregnancy stimulate normal prgnant women's neturophils for 2-4 hours.Quant-iTTM PicoGreen(?)dsDNA Reagent and Kits and live-cell fluorescence staining were used to detect the release of NETs7.Role of ROS release in antiphospholipid antibody-induced NETs releaseDCFH-DA reactive oxygen species assay kit and fluorescence microplate reader were used to detect the effect of antiphospholipid antibodies on ROS release from neutrophilsNeutrophils were pre-treated with NADPH oxidase inhibitor DPI for 30 minutes,and then stimulated with antiphospholipid antibodies for 2-4 hours.Quant-iTTM PicoGreen(?)dsDNA Reagent and Kits and live-cell staining were used to detect the release of NETs to observe the effect of ROS on NETs release.8.Effect of phosphorylation of Akt,ERK,p38-MAPK on antiphospholipid antibody-induced NETs formationWestern blot was used to detect the effects of antiphospholipid antibodies on the phosphorylation levels of Akt,ERK and p38-MAPK.Neutrophils were pre-treated by Akt,ERK,p38-MAPK phosphorylation inhibitors for 30 minutes,then were stimulated with antiphospholipid antibodies for 2-4 hours.Quant-iTrM PicoGreen(?)dsDNA Reagent and Kits and live-cell staining were used to detect the NETs release to observe the effects of the phosphorylation of Akt,ERK,p38-MAPK on the release of NETs.9.In vitro NETs isolationAntiphospholipid antibodies,normal control IgG or PMA were used to stimulate neutrophils for 4 hours,and then NETs were collected by centrifugation and vigorous shaking.10.Effects of antiphospholipid-induced NETs on trophoblast migration and invasionTrophoblasts were treated with APS-IgG-induced NETs for 12-24 hours.Transwell migration test and scratch test were used to detect trophoblast migration ability.Transwell invasion assay was used to detect trophoblast invasion.11.Effects of antiphospholipid-induced NETs on endothelial cell migration and angiogenesisHuman umbilical vein endothelial cells(HUVECs)were treated with APS-IgG?induced NETs for 12-24 hours.Transwell migration assay was used to detect endothelial cell migration,and angiogenesis assay was used to detect endothelial cell angiogenesis capabilities.Results1.Sera from pregnant women with OAPS show increased cell-free DNA and NETsCompared with healthy controls,APS sera had significantly greater levels of cell-free DNA.We further collected sera from 15 aPL-positive patients without pregnancy morbidities and 18 aPL-negative patients with pregnancy complications to determine cell-free DNA levels.aPL-positive patients with pregnancy morbidities showed significantly higher cell-free DNA levels than those of the other two groups.There are also higher levels of MPO-DNA complexes,MPO,and NE-credible markers of NETs-in APS sera than in HC sera.2.Neutrophils from pregnant patients with APS are primed for NETosisBoth the results of cell-free DNA detection and live-cell staining showed that without specific in vitro stimulation,APS neutrophils displayed intensified spontaneous NET release compared with that of HC neutrophils3.IgG from APS sera stimulates neutrophils to release NETsHC neutrophils were incubated with IgG isolated from APS sera(APS-IgG),HC sera(HC-IgG),and PMA.Neutrophils incubated with APS-IgG released more cell-free DNA than did neutrophils incubated with HC-IgG,which was confirmed by SYTOX Green staining and cell immunofluorescence.Therefore,APS-IgG significantly stimulates NET release compared with that of HC-IgG.4.Antiphospholipid antibodies increase NETs production by promoting ROS releaseNeutrophils from HCs were treated with DCFH-DA after stimulation with APS-IgG,HC-IgG,or PMA to analyze ROS production.APS-IgG accelerated ROS production in neutrophils in a time-dependent manner and showed a similar effect to that of PMA.This process was partialy depend on NADPH oxidase Pre-treatment of neutrophils with NADPH oxidase inhibitor DPI significantly decreased cell-free DNA levels,which was confirmed by live-cell imaging,highlighting the influence of ROS on these reactions5.Antiphospholipid antibodies promote NETs release by promoting phosphorylation of Akt,ERK,p38-MAPKNeutrophils from HCs were stimulated with APS-IgG,HC-IgG,or PMA.Western blot results showed that antiphospholipid antibodies promoted phosphorylation of Akt,ERK,and p38-MAPK.Pre-treatment of neutrophils with Akt,ERK,and p38-MAPK phosphorylation inhibitors can inhibit the release of NETs caused by antiphospholipid antibodies6.NETs triggered by APS-IgG play a detrimental role in the invasion and migration of trophoblasts and in the migration and angiogenesis capacity of HUVECsAntiphospholipid antibody-induced NETs treated early pregnancy extravillous trophoblast cell line HTR-8/Svneo.The results of transwell migration assay and wound-healing scratch assay showed that trophoblast migration ability was reduced Transwell invasion assay results showed that trophoblast invasion ability was decreased.Antiphospholipid antibody-induced NETs treated human umbilical vein endothelial cells(HUVECs).Transwell migration results showed endothelial cell migration capacity was reduced.And results of tube formation assay showed reduced angiogenesis capacity of HUVECsConclusionThe levels of NETs increase in the sera of early pregnancy patients with OAPS.Antiphospholipid antibodies induce the release of NETs by promoting ROS production and phosphorylation of Akt,ERK,p38-MAPK.Antiphospholipid antibodies-induced NETs impair the migration and invasion of trophoblasts as well as the migration and angiogenesis of endothelial cells,which in turn leads to pregnancy morbidity.